Replication infidelity via a mismatch with Watson-Crick geometry

Abstract

In describing the DNA double helix, Watson and Crick suggested that "spontaneous mutation may be due to a base occasionally occurring in one of its less likely tautomeric forms." Indeed, among many mispairing possibilities, either tautomerization or ionization of bases might allow a DNA polymerase to insert a mismatch with correct Watson-Crick geometry. However, despite substantial progress in understanding the structural basis of error prevention during polymerization, no DNA polymerase has yet been shown to form a natural base-base mismatch with Watson-Crick-like geometry. Here we provide such evidence, in the form of a crystal structure of a human DNA polymerase λ variant poised to misinsert dGTP opposite a template T. All atoms needed for catalysis are present at the active site and in positions that overlay with those for a correct base pair. The mismatch has Watson-Crick geometry consistent with a tautomeric or ionized base pair, with the pH dependence of misinsertion consistent with the latter. The results support the original idea that a base substitution can originate from a mismatch having Watson-Crick geometry, and they suggest a common catalytic mechanism for inserting a correct and an incorrect nucleotide. A second structure indicates that after misinsertion, the now primer-terminal G • T mismatch is also poised for catalysis but in the wobble conformation seen in other studies, indicating the dynamic nature of the pathway required to create a mismatch in fully duplex DNA.

Fig. 1.

Superposition of Pol λ DL ternary complexes. The complex with a dGMPCPP•T nascent mispair [the protein is green, the primer strand is yellow, the template strand is orange, the incoming dGMPCPP is magenta, and the active site metal ions (Me) are bright green] is overlaid with the ddTTP•A nascent base pair-containing complex, PDB ID code 3MGI (the protein is light cyan, the primer strand is beige, the template strand is light brown, and the incoming ddTTP is dark brown).

Fig. 2.

Watson–Crick conformation of a dGMPCPP•T mispair at the active site. (A) Superposition of the ternary complexes of Pol λ DL (green) in complex with DNA (primer strand is olive, template strand is orange) and incoming dGMPCPP (magenta) opposite template T (orange) and the WT Pol λ ternary complex (2PFO) (protein is gray, template strand is light orange, incoming dUMPNPP is light purple, and template adenine is brown. The metal ions A and B (Me) in the complex of Pol λ DL and WT Pol λ are green and purple, respectively. (B) Close up of the active site of the ternary complex of Pol λ DL wih the dGMPCPP•T nascent mispair. The magnesium ions are green, and active site H₂O molecules are light red.

Fig. 4.

Geometry of G•T mispairs in Pol λ DL complexes. (A) Watson–Crick conformation of dGMPCPP•T nascent base pair from the ternary, precatalytic complex. (B) Correct dGMPCPP•C nascent base pair from the precatalytic complex with the G•T primer–terminal base pair. (C) G•T primer–terminal base pair in wobble conformation in the precatalytic, ternary complex. Simulated annealing Fobs-Fcalc omit maps contoured at 3.5σ are shown in dark blue. (D) Base pair parameters (derived using 3DNA software v.1.5, Lu and Olson), including H-bond information [atom, pair, and length (Å)], base pair width [C1′—C1′ distance (Å)], and l_R and l_Y angles (in degrees) between the line joining the C1′—C1′ and the N9-C1′ (purine) and N1-C1′ (pyrimidine) glycosidic bonds. The positions of atoms in a G•C base pair are indicated.

Fig. 3.

G•T terminal mispair in the ternry complex of Pol λ DL. (A) Superposition of Pol λ DL ternary complex with a G•T terminal mispair and the WT Pol λ complex (2PFO). Pol λ DL is light gray, the template and primer strands are gray, and the G•T terminal mispair is bright green; the incoming dGMPCPP is magenta, and the active site metal ions A and B are purple and pea green, respectively. In the WT Pol λ complex, the protein is light blue, the DNA is light yellow, the incoming dUMPNPP is pink, and the metal ions A and B are light purple and dark green, respectively. (B) Close up of the active site of Pol λ DL ternary complex with a G•T terminal mispair.